Peptidylglycine-alpha-hydroxylating monooxygenase generates two hydroxylated products from its mechanism-based suicide substrate, 4-phenyl-3-butenoicacid

The bifunctional enzyme peptidylglycine-alpha-amidating monooxygenase media tes the conversion of C-terminal glycine-extended peptides to their active alpha-amidated products. Peptidylglycine-alpha-hydroxylating monooxygenase (PHM, EC 1.14. 17.3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent ir reversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PEA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PEA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PEA are metabolized per molecule of PHM inactivated, under saturating condition s. The metabolism of PEA by PHM generates two hydroxylated products, 2-hydr oxy-4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-b utenoic acid. While one enantiomer for each product is significantly favore d in the reaction, both are produced. From these observations, we conclude that hydroxylated PEA products are formed by a delocalized free radical mec hanism and that the lack of absolute stereospecificity indicates significan t freedom of movement within the catalytic site, The ability of PHM to meta bolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines.